JP6280180B2 - Methods and compositions for modulating angiogenesis and vascular development - Google Patents

Description

This application is related to US Provisional Patent Application No. 61 / 591,486, filed Jan. 27, 2012, and US Provisional Patent Application No. 61/637, filed Apr. 24, 2012. No. 740, US Provisional Patent Application No. 61 / 709,619, filed Oct. 4, 2012 (the specification and drawings are incorporated herein by reference in their entirety for all purposes) Insist on the benefits).

Statement on federal support Not applicable.

  The present invention relates to the field of angiogenesis and vascular development for the treatment of ischemic events such as, for example, stroke. The present invention also relates to the field of stem cells and stem cells derived by genetic manipulation.

  In stable stroke, restoration of blood flow is essential to restore nutrient supply in the brain. To repair vascular damage after prolonged ischemia, at least two successive steps are required. The first step is angiogenic sprouting of endothelial cells (EC); this process involves initial proliferation of endothelial cells and remodeling of the surrounding extracellular matrix. EC VEGF-mediated growth and matrix metalloproteinases are one of the major components of angiogenic germination. The second step is vascular stabilization; a process that relies on the recruitment of vascular smooth muscle cells to wrap young blood vessels. Monocytes and pericytes are also involved in vascular stabilization, production of appropriate arterial factors and extracellular matrix proteins. In the absence of vascular stabilization by smooth muscle cells and pericytes, regression of the new vasculature can occur.

  Bone marrow stromal cells (also known as MSCs, also known as mesenchymal stem cells) have been shown to promote vascular regeneration following cerebral artery occlusion and traumatic brain injury (Omori et al. (2008) Brain Res. 1236: 30.38; Onda et al. (2008) J. Cereb.Blood Flow Metab.28: 329.340; Pavlicenko et al. (2008) Brain Res.1233: 203.213; Xiong et al. (2009) Brain Res.・ 191). SB623 cells are derivatives of bone marrow stromal cells and are obtained by transfecting bone marrow stromal cells with a vector containing a sequence encoding the Notch intracellular domain (NICD). For example, US Pat. No. 7,682,825 and Dezawa et al. (2004) J. MoI. Clin. Investig. 13: 1701-1710. SB623 cells produce functional improvements in an experimental stroke model. See, for example, US Pat. No. 8,092,792 and Yashara et al. (2009) Stem Cells and Development 18: 1501-1514. Although the secretome of SB623 cells is comparable to that of the parent MSC, it is observed that different levels of specific trophic factors are secreted by MSC compared to SB623 cells. See, for example, Tate et al. (2010) Cell Transplantation 19: 973-984; U.S. Patent Application Publication No. 2010/0266554. In addition, a number of factors that have different expression levels between MSC and SB623 cells have been reported to be involved in revascularization.

  Because stroke is the leading cause of adult disability in the United States and the second leading cause of death worldwide, it restores blood supply to the area of stroke-induced ischemic injury in the brain, and There remains a need for treatment to promote perfusion.

  We are astonishing that the progeny of mesenchymal stem cells transfected with sequences encoding the Notch intracellular domain (ie, SB623 cells) can synthesize and secrete factors that promote angiogenesis. It has been found that it has properties. Since angiogenesis, ie the formation of new blood vessels, is an important part of the endogenous repair process in brain injury and disease, this discovery provides a new treatment for vascular disorders such as stroke.

Accordingly, the present disclosure provides among others:
1. A method for increasing angiogenesis in a subject, the method comprising administering to the subject a population of SB623 cells; wherein the SB623 cells are (a) a culture of mesenchymal stem cells. And (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length Notch protein. , (C) selecting a cell comprising the polynucleotide of step (b), and (d) a method obtained by further culturing the selected cell of step (c) without selection.

  2. The method of embodiment 1, wherein the increased angiogenesis occurs in the central nervous system.

  3. The method of embodiment 2, wherein the increased angiogenesis occurs in the brain.

  4). A method for repairing ischemic damage in a subject comprising administering to said subject a population of SB623 cells; wherein said SB623 cells are (a) cultured mesenchymal stem cells And (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length Notch protein. And (c) selecting a cell comprising the polynucleotide of step (b), and (d) a method obtained by further culturing the selected cell of step (c) without selection.

  5. Embodiment 5. The method of embodiment 4, wherein the ischemic damage occurs in the central nervous system.

  6). Embodiment 6. The method of embodiment 5, wherein the ischemic injury occurs in the brain.

  7). Embodiment 7. The method of embodiment 6, wherein the ischemic injury is due to a stroke.

  8). A method for enhancing the survival of endothelial cells, said method comprising contacting said endothelial cells with a population of SB623 cells; wherein said SB623 cells are (a) cultured mesenchymal stem cells And (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length Notch protein. And (c) selecting a cell comprising the polynucleotide of step (b), and (d) a method obtained by further culturing the selected cell of step (c) without selection.

  9. Embodiment 9. The method of embodiment 8, wherein the method prevents endothelial cell death.

  10. Embodiment 10. The method of embodiment 8 or 9, wherein the endothelial cell is in a subject.

  11. Embodiment 11. The method of embodiment 10, wherein the endothelial cells are in the central nervous system of the subject.

  12 12. The method of embodiment 11, wherein the endothelial cells are in the subject's brain.

  13. A method for stimulating endothelial cell proliferation, comprising contacting the endothelial cell with a population of SB623 cells; wherein the SB623 cell comprises (a) a mesenchymal stem cell Preparing a culture, (b) a cell culture of step (a) and a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein said polynucleotide does not encode the full length of a Notch protein. A method obtained by contacting, (c) selecting cells comprising the polynucleotide of step (b), and (d) further culturing the selected cells of step (c) without selection.

  14 14. The method of embodiment 13, wherein the endothelial cell is in a subject.

  15. Embodiment 15. The method of embodiment 14, wherein the endothelial cells are in the central nervous system of the subject.

  16. Embodiment 16. The method of embodiment 15, wherein the endothelial cells are in the subject's brain.

  17. A method for promoting vascular branching comprising contacting the blood vessel with a population of SB623 cells; wherein the SB623 cells are (a) a culture of mesenchymal stem cells And (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length Notch protein. , (C) selecting a cell comprising the polynucleotide of step (b), and (d) a method obtained by further culturing the selected cell of step (c) without selection.

  18. Embodiment 18. The method of embodiment 17, wherein the blood vessel is in a subject.

  19. Embodiment 19. The method of embodiment 18, wherein the blood vessel is in the central nervous system of the subject.

  20. 20. The method of embodiment 19, wherein the blood vessel is in the subject's brain.

21. A method for increasing angiogenesis in a subject, said method comprising:
(1) A population of SB623 cells; wherein the SB623 cells prepare (a) a culture of mesenchymal stem cells, (b) the cell culture of step (a), and a Notch intracellular domain (NICD) Contacting a polynucleotide comprising a sequence encoding, wherein said polynucleotide does not encode the full length of a Notch protein, (c) selecting a cell comprising the polynucleotide of step (b), and (d) step ( c) obtained by further culturing the selected cells without selection; and (2) administering a pro-angiogenic agent.

  22. Embodiment 22. The method of embodiment 21, wherein the increased angiogenesis occurs in the central nervous system.

  23. 24. The method of embodiment 22, wherein the increased angiogenesis occurs in the brain.

  24. The method of embodiment 21, wherein the angiogenesis promoting agent is a nucleic acid.

  25. The method according to embodiment 21, wherein the angiogenesis-promoting agent is a polypeptide.

  26. 26. The method of embodiment 25, wherein the polypeptide is a transcription factor that activates expression of a pro-angiogenic protein.

  27. 27. The method of embodiment 26, wherein the pro-angiogenic protein is vascular endothelial growth factor (VEGF).

  28. 28. The method of embodiment 27, wherein the transcription factor is a non-natural zinc finger protein that activates transcription of the VEGF gene.

29. A method for repairing ischemic damage in a subject, said method comprising:
(1) A population of SB623 cells; wherein the SB623 cells prepare (a) a culture of mesenchymal stem cells, (b) the cell culture of step (a), and a Notch intracellular domain (NICD) Contacting a polynucleotide comprising a sequence encoding, wherein said polynucleotide does not encode the full length of a Notch protein, (c) selecting a cell comprising the polynucleotide of step (b), and (d) step ( c) obtained by further culturing the selected cells without selection; and (2) administering a pro-angiogenic agent.

  30. 30. The method of embodiment 29, wherein the ischemic damage occurs in the central nervous system.

  31. 32. The method of embodiment 30, wherein the ischemic injury occurs in the brain.

  32. 32. The method of embodiment 31, wherein the ischemic injury is due to a stroke.

  33. 30. The method of embodiment 29, wherein the angiogenesis promoting agent is a nucleic acid.

  34. 30. The method of embodiment 29, wherein the angiogenesis promoting agent is a polypeptide.

  35. 35. The method of embodiment 34, wherein the polypeptide is a transcription factor that activates expression of a pro-angiogenic protein.

  36. 36. The method of embodiment 35, wherein the pro-angiogenic protein is vascular endothelial growth factor (VEGF).

  37. 37. The method of embodiment 36, wherein the transcription factor is a non-natural zinc finger protein that activates transcription of the VEGF gene.

38. A method for treating a stroke in a subject, said method comprising:
(1) A population of SB623 cells; wherein the SB623 cells prepare (a) a culture of mesenchymal stem cells, (b) the cell culture of step (a), and a Notch intracellular domain (NICD) Contacting a polynucleotide comprising a sequence encoding, wherein said polynucleotide does not encode the full length of a Notch protein, (c) selecting a cell comprising the polynucleotide of step (b), and (d) step ( c) obtained by further culturing the selected cells without selection; and (2) administering a pro-angiogenic agent.

  39. 39. The method of embodiment 38, wherein the angiogenesis promoting agent is a nucleic acid.

  40. 39. The method of embodiment 38, wherein the pro-angiogenic agent is a polypeptide.

  41. 41. The method of embodiment 40, wherein the polypeptide is a transcription factor that activates expression of a pro-angiogenic protein.

  42. 42. The method of embodiment 41, wherein the pro-angiogenic protein is vascular endothelial growth factor (VEGF).

  43. 43. The method of embodiment 42, wherein the transcription factor is a non-natural zinc finger protein that activates transcription of the VEGF gene.

  44. Embodiment 14. The method of embodiment 8, 9 or 13, further comprising administering an angiogenesis promoting agent in conjunction with the SB623 cells.

  45. 45. The method of embodiment 44, wherein the endothelial cells are in a subject.

  46. 46. The method of embodiment 45, wherein the endothelial cells are in the central nervous system of the subject.

  47. 47. The method of embodiment 46, wherein the endothelial cells are in the subject's brain.

  48. 45. The method of embodiment 44, wherein the angiogenesis promoting agent is a nucleic acid.

  49. 45. The method of claim 44, wherein the pro-angiogenic agent is a polypeptide.

  50. 50. The method of embodiment 49, wherein the polypeptide is a transcription factor that activates expression of a pro-angiogenic protein.

  51. 51. The method of embodiment 50, wherein the pro-angiogenic protein is vascular endothelial growth factor (VEGF).

  52. 52. The method of embodiment 51, wherein the transcription factor is a non-natural zinc finger protein that activates transcription of the VEGF gene.

  53. 18. The method of embodiment 17, further comprising administering a pro-angiogenic agent along with the SB623 cells.

  54. 54. The method of embodiment 53, wherein the blood vessel is in a subject.

  55. 55. The method of embodiment 54, wherein the blood vessel is in the central nervous system of the subject.

  56. 56. The method of embodiment 55, wherein the blood vessel is in the subject's brain.

  57. 54. The method of embodiment 53, wherein the angiogenesis promoting agent is a nucleic acid.

  58. 54. The method of embodiment 53, wherein the pro-angiogenic agent is a polypeptide.

59. 59. The method of embodiment 58, wherein the polypeptide is a transcription factor that activates expression of a pro-angiogenic protein.
60. 60. The method of embodiment 59, wherein the pro-angiogenic protein is vascular endothelial growth factor (VEGF).

  61. 61. The method of embodiment 60, wherein the transcription factor is a non-natural zinc finger protein that activates transcription of the VEGF gene.

  62. A method for providing an angiogenic factor to a subject, wherein said method comprises administering to said subject a population of SB623 cells; wherein said SB623 cells are (a) mesenchymal Preparing a culture of stem cells, (b) a polynucleotide comprising the cell culture of step (a) and a sequence encoding a Notch intracellular domain (NICD), wherein said polynucleotide does not encode the full length of the Notch protein And (c) selecting cells containing the polynucleotide of step (b), and (d) obtaining the selected cells of step (c) without further selection.

  63. 64. The method of embodiment 62, wherein the subject is suffering from an ischemic disorder.

  64. 64. The method of embodiment 63, wherein the subject is suffering from a central nervous system disease or disorder.

  65. The trophic factors include angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin-binding epidermal growth factor-like growth factor, hepatocyte growth factor, leptin, platelet derived growth factor-BB, placental growth factor, 63. The method of embodiment 62, wherein the method is one or more selected from the group consisting of: and vascular endothelial growth factor.

  66. 66. The method of embodiment 65, wherein the trophic factor is vascular endothelial growth factor.

  67. A method for providing vascular endothelial growth factor to a subject, wherein said method comprises administering a population of SB623 cells to said subject; wherein said SB623 cells are between (a) Preparing a culture of mesenchymal stem cells; (b) a cell culture of step (a) and a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein said polynucleotide comprises the full length Notch protein; A non-coding contact, obtained by (c) selecting cells comprising the polynucleotide of step (b), and (d) further culturing the selected cells of step (c) without selection .

  68. 68. The method of embodiment 67, wherein the subject is an ischemic disorder.

  69. 69. The method of embodiment 68, wherein the subject is suffering from a central nervous system disease or disorder.

FIG. 1 shows the measurement of the fraction of cells that are permeable to propidium iodide in cultures of HUVEC that are deficient in serum and growth factors. The leftmost bar shows the results obtained from the control serum / growth factor deficient HUVEC, the middle bar shows the results of the serum / growth factor deficient HUVEC cultured for 7 days in the presence of MSC-derived conditioned medium, and The far right bar shows the results of serum / growth factor deficient HUVECs cultured for 7 days in the presence of conditioned medium from SB623 cells. Values indicate mean ± SD for 3 independent donors of MSC and SB623 cells ( ^* indicates p <0.05 compared to the control group). FIG. 2 shows the measurement of the cell fraction expressing Bcl-2 in cultures of HUVEC depleted of serum and growth factors. The leftmost bar shows the results obtained from the control serum / growth factor deficient HUVEC, the middle bar shows the results of the serum / growth factor deficient HUVEC cultured for 7 days in the presence of MSC-derived conditioned medium, and The far right bar shows the results of serum / growth factor deficient HUVECs cultured for 7 days in the presence of conditioned medium from SB623 cells. Results were obtained by measuring the fluorescence of cells stained with fluorescein-conjugated anti-Bcl-2 antibody and subtracting the fluorescence of cells subjected to fluorescein-conjugated IgG. Values indicate mean ± SD for 3 independent donors of MSC and SB623 cells ( ^* indicates p <0.05 compared to control group). FIG. 3 shows the measurement of the fraction of cells expressing Ki67 in cultures of HUVEC depleted of serum and growth factors. The leftmost bar shows the results obtained from the control serum / growth factor deficient HUVEC, the middle bar shows the results of the serum / growth factor deficient HUVEC cultured for 7 days in the presence of MSC-derived conditioned medium, and The far right bar shows the results of serum / growth factor deficient HUVECs cultured for 7 days in the presence of conditioned medium from SB623 cells. Results were obtained by measuring the fluorescence of cells stained with fluorescein-conjugated anti-Ki67 antibody and subtracting the fluorescence of cells subjected to fluorescein-conjugated IgG. Values indicate mean ± SD for 3 independent donors of MSC and SB623 cells ( ^* indicates p <0.05 compared to control group). FIG. 4 shows a photomicrograph of a HUVEC phase contrast microscope after 16 hours of culture in conditioned medium from MSC or SB623 cells. The leftmost photo shows cells cultured in conditioned medium from MSC, the middle photo shows cells cultured in conditioned medium from SB623 cells, and the rightmost photo in a commercial medium without the addition of conditioned medium. Cultured cells are shown. FIG. 5 shows a measurement of the effect of conditioned medium on tube formation by HUVEC. The leftmost bar shows the results obtained from the control serum / growth factor deficient HUVEC, the middle bar shows the results of the serum / growth factor deficient HUVECs cultured for 16 hours in the presence of MSC-derived conditioned medium, and The far right bar shows the results of serum / growth factor deficient HUVECs cultured for 16 hours in the presence of conditioned medium from SB623 cells. Values represent the mean ± SEM for 3 independent donors of MSC and SB623 cells. FIG. 6 shows a photograph of the aortic ring after 10 days of culture in non-conditioned medium (FIG. 6A), MSC conditioned medium (FIG. 6B), or SB623 cell conditioned medium (FIG. 6C). FIG. 7A shows the measurement of vascular sprouting and branching in the aortic ring assay. FIG. 7A shows the new vessel and the total number of branch points in the new vessel. For each of the three pairs of bars, the left bar shows the measurement of new angiogenesis and the right bar shows the measurement of vessel branching. The leftmost bar pair ("Subject") shows the results obtained from the control aortic ring, and the middle bar pair ("MSC CM") was obtained from the aortic ring cultured in MSC conditioned medium for 10 days. Results are shown, and the rightmost bar pair (“SB623 CM”) shows results obtained from aortic rings cultured in SB623 cell conditioned medium for 10 days. FIG. 7B shows the measurement of vascular sprouting and branching in the aortic ring assay. FIG. 7B shows the branch points to new blood vessels for the control aortic ring (left bar), the ring cultured for 10 days in MSC conditioned medium (middle bar) and the ring cultured for 10 days in SB623 cell conditioned medium (right bar). Indicates the percentage. Values represent mean ± SEM for 7 donor pairs ( ^* indicates p <0.05 compared to control group). FIG. 8 shows the levels of four different trophic factors in conditioned media from MSC (light bar) and SB623 cells (dark bar). Protein levels are shown as picograms / ml (10 ⁶ cells per conditioned medium). As shown, MSC-derived conditioned media (and SB623 cells derived from them) from 4 human donors were tested. The levels of angiogenin, angiopoietin-2, heparin-binding epidermal growth factor-like growth factor (HB-EGF), and placental growth factor (PIGF) are shown. FIG. 9 shows the levels of 10 different cytokines in conditioned media from MSC and SB623 cells. Cells for the production of conditioned media were obtained from four different donors (D1, D2, D3 and D4) as shown in the figure. The figure reveals the enormous amount of VEGF produced by MSC and SB623 cells compared to the levels of other trophic factors tested. Abbreviations are shown in the legend of Table 1 (Example 6). FIG. 10A shows the effect of VEGF receptor inhibitors on the improvement in HUVEC viability enhanced by SB623 cell conditioned media. FIG. 10A shows the cell fraction permeable to propidium iodide in cultures of HUVEC lacking serum and growth factors. The leftmost bar shows the results obtained from control serum / growth factor deficient HUVECs, the middle bar shows the results of serum / growth factor deficient HUVECs cultured for 5 days in the presence of conditioned media from SB623 cells, The rightmost bar shows the results of serum / growth factor-deficient HUVECs cultured for 5 days in the presence of conditioned medium derived from SB623 cells and 50 nM SU5416. Results are averages from two donors (“ ^* ” indicates p <0.05 versus control medium, “#” versus SB623 cell conditioned medium and medium subjected to SU5416. p <0.05 is indicated). FIG. 10B shows the effect of VEGF receptor inhibitors on the improvement in HUVEC viability enhanced by SB623 cell conditioned media. FIG. 10B shows the measurement of the cell fraction expressing Bcl-2 in cultures of HUVEC lacking serum and growth factors. The leftmost bar shows the results obtained from control serum / growth factor deficient HUVECs, the middle bar shows the results of serum / growth factor deficient HUVECs cultured for 5 days in the presence of conditioned media from SB623 cells, The rightmost bar shows the results of serum / growth factor-deficient HUVECs cultured for 5 days in the presence of conditioned medium derived from SB623 cells and 50 nM SU5416. Results are averages from duplicate donors and were obtained by measuring the fluorescence of cells stained with fluorescein-conjugated anti-Bcl-2 antibody and subtracting the fluorescence of cells subjected to fluorescein-conjugated IgG. FIG. 11 shows measurement of the fraction of cells expressing Ki67 in HUVEC cultures subjected to SB623 cell conditioned medium in the presence and absence of VEGFR2 inhibitor SU5416 and control cells cultured in the absence of CM. . The leftmost bar (clear) shows the results obtained from the control serum / growth factor deficient HUVEC, and the middle bar (black) is of serum / growth factor deficient HUVEC cultured in the presence of conditioned medium from SB623 cells. Results are shown, and the rightmost bar (gray) shows the results of serum / growth factor deficient HUVECs cultured in the presence of conditioned media from SB623 cells and 50 nM SU5416. Values indicate mean ± SEM for two independent donors of SB623 cells (“ ^* ” indicates p <0.05 vs. negative control cultures (no conditioned media); “#” indicates SU5416 treated cultures) P <0.05 relative to product). FIG. 12 shows the effect of VEGF receptor inhibitors on the promotion of tube formation by HUVEC enhanced by MSC and SB623 cell conditioned media. The top row shows cells cultured in the absence of inhibitor. The left end of the upper row (“Neg”) shows a phase contrast micrograph of a control HUVEC after 16 hours in Opti-MEM medium. The second panel from the left (“+10 ng VEGF”) shows a phase contrast micrograph of HUVEC after 16 hours of culturing in Opti-MEM medium supplemented with 10 ng / ml VEGF. The third panel from the left (“+ MSC-CM”) shows a phase contrast micrograph of HUVEC after 16 hours of culture in MSC conditioned medium. The rightmost panel (“+ SB623-CM”) shows a phase contrast micrograph of HUVEC after 16 hours of culture in SB623 cell conditioned medium. The bottom row panel shows a photomicrograph of HUVEC under the same conditions as the top row, but with the addition of 50 nM SU5416. FIG. 13 shows quantification of tube formation by HUVEC subjected to SB623 cell conditioned medium in the presence and absence of VEGFR2 inhibitor SU5416, and control cells cultured in the absence of CM. For each time point, the leftmost bar (clear) shows the results obtained from the control serum / growth factor deficient HUVEC and the middle bar (black) is the serum / cultured in the presence of conditioned medium from SB623 cells. Results for growth factor deficient HUVEC are shown, and the rightmost bar (gray) shows results for serum / growth factor deficient HUVEC cultured in the presence of SB623 cell-derived conditioned medium and 50 nM SU5416. Values indicate mean ± SEM for three independent donors of SB623 cells (“*” indicates p <0.05 vs. negative control cultures (no conditioned media), “#” indicates SU5416-treated) P <0.05 for culture). Figure 14 shows the effect of VEGF receptor inhibitors on the promotion of vascular outgrowth and branching enhanced by SB623 cell conditioned medium in the aortic ring assay. In the upper row, the left panel shows a micrograph of the aortic ring after 10 days culture on RGF basement gel in OptiMEM medium (“negative control”). The middle panel shows a micrograph of the aortic ring after 10 days in SB623 cell conditioned medium (“+ SB623-CM”). The right panel shows a micrograph of the aortic ring after 10 days culture in SB623 cell conditioned medium containing 50 nM SU5416 ("+ SB623-CM + SU5416"). An enlargement of a specific area of each micrograph is shown in the bottom row.

  The present specification discloses novel methods and compositions for modulation of angiogenesis. In particular, factors secreted by SB623 cells (MSC-derived progeny cells transfected with vectors containing sequences encoding the Notch intracellular domain) promote endothelial cell survival and proliferation in vitro under serum and growth factor deficient conditions. Promotes and stimulates tube formation by human umbilical vein endothelial cells. In addition, conditioned medium derived from SB623 cells promotes endothelial sprouting and branching in the rodent aortic ring assay.

Implementation of the present disclosure is within the fields of cell biology, toxicology, molecular biology, biochemistry, cell culture, immunology, oncology, recombinant DNA and techniques in the art unless otherwise noted. Utilize standard methods and conventional techniques in related fields. Such techniques are described in the literature and are therefore available to those skilled in the art. For example, Alberts, B .; Et al., "Molecular Biology of the Cell, " 5 th edition, Garland Science, New York, NY, 2008; Voet, D. Et al., "Fundamentals of Biochemistry: Life at the Molecular Level," 3 rd edition, John Wiley & Sons, Hoboken, NJ, 2008; Sambrook, J. Et al, "Molecular Cloning: A Laboratory Manual ," 3 rd edition, Cold Spring Harbor Laboratory Press, 2001; Ausubel, F. Et al., "Current Protocols in Molecular Biology," John Wiley & Sons, New York, 1987 and periodic updates; I. , "Culture of Animal Cells: A Manual of Basic Technique," 4 th edition, John Wiley & Sons, Somerset, NJ, 2000; and "Methods in Enzymology" series, Academic Press, San Diego, see the CA.

  For purposes of this disclosure, “angiogenesis” refers to the formation of new vasculature (eg, blood vessels; eg, veins, arteries, venules, arterioles, capillaries). Angiogenesis occurs by the sprouting of new blood vessels from existing blood vessels and / or branching of blood vessels. Angiogenesis also includes the processes associated with matrix remodeling and cell mobilization (eg, smooth muscle cell, monocyte and / or pericyte cell mobilization).

  “MSC” means adherent, non-hematopoietic stem cells obtained from bone marrow. These cells are variously known as mesenchymal stem cells, mesenchymal stromal cells, bone marrow adherent stromal cells, bone marrow adherent stem cells, and bone marrow stromal cells.

Stroke “Stroke” is the name given to a condition resulting from impaired blood flow in the brain. Such cerebral vascular dysfunction may result from, for example, intracranial hemorrhage, or worsening or obstruction of blood flow in the brain (ie, cerebral ischemia). Ischemic occlusion can be due to thrombosis (ie, the formation of a clot at that location in the cranial or blood supply to the brain) or cerebral embolism (ie, movement of the clot to a location in the brain). Injury resulting from ischemia or hemorrhagic stroke usually leads to impairment of neurological function. Further information regarding the different types of strokes and their characteristics can be found in sharing US Pat. No. 8,092,792; the disclosure of which is provided herein for purposes of describing the different types of strokes and their characteristics. The entirety of which is incorporated by reference.

Mesenchymal stem cells (MSC)
The present disclosure relates to a method for promoting angiogenesis by transplanting SB623 cells at a site of ischemic injury in a subject. SB623 cells are obtained from expressing the intracellular domain of Notch protein in MSCs, also known as bone marrow adherent stromal cells, also known as mesenchymal stem cells (MSCs). MSCs are obtained by selecting adherent cells (ie, cells that adhere to tissue culture plastic) from the bone marrow.

  Exemplary disclosures of MSCs are provided in U.S. Patent Application Publication No. 2003/0003090; Science 276: 71-74 and Jiang (2002) Nature 418: 41-49. Methods for isolation and purification of MSC are described, for example, in US Pat. No. 5,486,359; Pittenger et al. (1999) Science 284: 143-147 and Dezawa et al. (2001) Eur. J. et al. Neurosci. 14: 1771-1776. Human MSCs are commercially available (eg, BioWhittaker, Walkersville, MD) or obtained from a donor, eg, by selection of bone marrow aspiration followed by adherent bone marrow cells. See, for example, International Publication No. 2005/100552.

  MSCs can be isolated from umbilical cord blood. See, for example, Campagnari et al. (2001) Blood 98: 2396-2402; Erics et al. (2000) Br. J. et al. Haematol. 109: 235-242 and Hou et al. (2003) Int. J. et al. Hematol. 78: 256-261.

Notch intracellular domain The Notch protein is a transmembrane receptor that affects cell differentiation through intracellular signaling found in all metazoans. Contact with Notch ligands of the Notch extracellular domain (eg, Delta, Serrate, Jagged) results in two proteolytic cleavages of the Notch protein, the second of which is caused by γ-secretase. Catalyzed and releases Notch intracellular domain (NICD) into the cytoplasm. In the mouse Notch protein, this cleavage occurs between amino acids gly 1743 and val 1744. NICD translocates to the nucleus where it acts as a transcription factor and recruits additional transcriptional regulatory proteins (eg, MAM, histone acetylase) to mitigate transcriptional repression of various target genes (eg, Hes1).

  Further details and information regarding Notch signaling can be found in, for example, Artavanis Tsakonas et al. (1995) Science 268: 225-232; Mumm and Kopan (2000) Developer. Biol. 228: 151-165 and Ehebauer et al. (2006) Sci. STKE 2006 (364), cm7. [DOI: 10.1126 / stke. 3642006 cm7].

Cell culture and transfection Standard methods for cell culture are known in the art. For example, R.A. I. See Freshney "Culture of Animal Cells: A Manual of Basic Technique," Fifth Edition, Wiley, New York, 2005.

  Methods for introducing foreign DNA into cells (ie, transfection) are also well known in the art. See, for example, Sambrook et al., “Molecular Cloning: A Laboratory Manual,“ Third Edition, Cold Spring Laboratory Press, 2001; Ausubel et al., “Current Protocol in Hoc. That.

SB623 cells In one embodiment for the preparation of SB623 cells, a culture of MSCs is contacted (eg, by transfection) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD); The transfected cells are enriched by selection and further cultured. For example, U.S. Patent No. 7,682,825 (March 23, 2010); U.S. Patent Application Publication No. 2010/0266554 (Oct. 21, 2010); and International Publication No. 2009/023251 (2009 2). 19th month; all of them disclose the isolation of mesenchymal stem cells and SB623 cells of mesenchymal stem cells (in those documents “neural precursor cells” and “neural regenerative cells”). for the purpose of describing the conversion to "representing a neural regenerating cell"), which are incorporated by reference in their entirety.

  In these methods, any polynucleotide (eg, vector) encoding a Notch intracellular domain can be used, and any method for selection and enrichment of transfected cells can be used. For example, in certain embodiments, the vector includes a sequence that encodes a Notch intracellular domain and is transfected with a vector that includes a sequence that encodes a drug resistance marker (eg, resistance to G418). In a further embodiment, two vectors (one containing a sequence encoding a Notch intracellular domain and one containing a drug resistance marker) are used for transfection of MSCs. In these embodiments, the selection is sufficient to cause the cell culture to kill insufficient cells containing the vector rather than cells that do not contain the vector after transfection of the cell culture with the vector (s). This is accomplished by adding an appropriate amount of a selective agent (eg, G418). Absence of selection requires removal of the selection agent or reduction of its concentration to a level that does not kill cells that do not contain the vector. Subsequent selection (eg, 7 days) removes the selected agent and the cells are further cultured (eg, passage 2).

  Preparation of SB623 cells thus involves transient expression of the foreign Notch intracellular domain in the MSC. To achieve this goal, MSCs can be transfected with a vector comprising a sequence encoding a Notch intracellular domain, wherein the sequence does not encode the full length of the Notch protein. All such sequences are well known and readily available to those skilled in the art. For example, Del Amo et al. (1993) Denomics 15: 259-264 shows the complete amino acid sequence of the mouse Notch protein; further Mumm and Kopan (2000) Devel. Biol. 228: 151-165 provides the amino acid sequence from the Notch protein that surrounds the so-called S3 cleavage site that releases the intracellular domain. Taken together, these references provide those of ordinary skill in the art with any peptide that includes a Notch intracellular domain that is not the full length of a Notch protein; thus, also provides those skilled in the art with a Notch intracellular domain that does not encode the full length of a Notch protein. All polynucleotides containing the encoding sequence are provided. Prior references (Del Amo and Mumm) are incorporated by reference in their entirety for the purpose of disclosing the full-length amino acid sequence of the Notch protein and the amino acid sequence of the Notch intracellular domain, respectively.

  Similar information is available for Notch proteins and nucleic acids from additional species, including rat, xenopus, drosophila, and human. For example, Weinmaster et al. (1991) Development 113: 199-205; Schroeter et al. (1998) Nature 393: 382-386; NCBI Reference SEQ ID NO: NM — 016167 (and references cited therein); SwissProt P46531 (and therein) See cited references); SwissProt Q01705 (and references cited therein); and GenBank CAB40733 (and references cited therein). Prior literature is incorporated by reference in their entirety for the purpose of disclosing the full-length amino acid sequence of the Notch protein and the amino acid sequence of the Notch intracellular domain in a number of different species.

  In a further embodiment, SB623 cells are prepared by introducing into the MSC a nucleic acid comprising a sequence encoding a Notch intracellular domain such that the MSC does not express a foreign Notch extracellular domain. It can be achieved, for example, by transfecting MSCs with a vector comprising a sequence encoding the Notch intracellular domain, wherein the sequence does not encode the full length of the Notch protein.

  For further details on the preparation of SB623 cells and methods for producing cells having characteristics similar to SB623 cells that can be used in the methods disclosed herein, see US Pat. No. 7,682,825; Publications 2010/0266554 (October 21, 2010) and 2011/0229442 (September 22, 2011); these disclosures provide further details and alternative methods for the preparation of SB623 cells; And is incorporated herein by reference for the purpose of providing a method for preparing cells having characteristics similar to SB623 cells. Also, Dezawa et al. (2004) J. MoI. Clin. Invest. 113: 1701-1710.

Uses As disclosed herein, the inventors have determined that progeny of mesenchymal stem cells (ie, SB623 cells) in which the Notch intracellular domain is transiently expressed have angiogenic activity; and It has been found that cells synthesize and secrete angiogenic factors. Thus, transplantation of SB623 cells is useful for the treatment of disorders whose therapeutic utility can be achieved by increasing angiogenesis in a subject. Such disorders include, but are not limited to, cerebral ischemia (eg, stroke), cardiac ischemia (eg, ischemic heart disease), intestinal ischemia (eg, ischemic colitis, mesenteric ischemia), extremities Ischemia, cutaneous ischemia, ischemic eye syndrome (eg, retinal ischemia) and cerebral palsy.

  Thus, as described herein, SB623 cells can be used in a number of methods related to stimulation of angiogenesis. These include, but are not limited to, treatment of any of the disorders mentioned in the previous paragraph, increased angiogenesis, repair of ischemic damage, prevention of endothelial cell death, improved endothelial cell survival, endothelial cell proliferation Stimulation and / or promotion of vessel branching may be mentioned.

  Such methods can be performed in vitro or in a subject. The subject can be a mammal, preferably a human. Stimulation of angiogenesis by SB623 cells as disclosed herein and the effects associated with such stimulation can occur, for example, in the central nervous system (eg, in the brain).

  Transplantation of SB623 cells can also be used in a method for supplying one or more angiogenic trophic factors to a subject. Such factors include, but are not limited to, angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin-binding epidermal growth factor-like growth factor, hepatocyte growth factor, leptin, platelet derived growth factor-BB , Placental growth factor, and vascular endothelial growth factor.

  In a further embodiment, SB623 cells may be used in combination with a second pro-angiogenic agent in combination therapy to increase angiogenesis in a subject. The combination therapy can be used for all the purposes described above. The second pro-angiogenic agent can be, for example, a small molecule drug, nucleic acid, or polypeptide (eg, antibody, transcription factor). Exemplary nucleic acids are triplex-forming nucleic acids, ribozymes and siRNA that activate the expression of angiogenic proteins and / or block the expression of anti-angiogenic proteins. Exemplary antibodies are antibodies that bind to angiogenic proteins (or other angiogenic agents) and / or inhibit the activity of angiogenic proteins (or other angiogenic agents). Exemplary transcription factors are those that inhibit transcription of a gene encoding one or more anti-angiogenic proteins, and those that activate transcription of one or more pro-angiogenic proteins. Anti-angiogenic and pro-angiogenic proteins are known in the art. Exemplary anti-angiogenic proteins include pigment epithelium derived factor (PEDF) and placental growth factor (PIGF). Exemplary pro-angiogenic proteins include vascular endothelial growth factor (VEGF), angiopoietin, and hepatocyte growth factor (HGF).

  In certain embodiments, the transcription factor as described above is a non-natural (artificial) transcription factor. An example of such a non-natural transcription factor is a non-natural zinc finger protein that has been modified to bind to a DNA sequence in cellular chromatin that regulates transcription of a target gene (eg, VEGF gene). The modified zinc finger transcription factor comprises a transcriptional regulatory domain (eg, a transcription activation domain or a transcription repression domain) in addition to the modified zinc finger DNA binding domain, as is known in the art.

  Methods for modifying zinc finger DNA binding domains to bind to selected DNA sequences are well known in the art. See, for example, Beerli et al. (2002) Nature Biotechnol, 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70: 313-340; Isalan et al. (2001) Nature Biotechnol. 19: 656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12: 632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10: 411-416. The zinc finger binding domain is modified to have a new binding specificity compared to the native zinc finger protein. Modification methods include, but are not limited to, selection methods based on rational design and various types of experiments. Rational designs include, for example, the use of a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is a specific triplet or quadruplet. Associated with one or more amino acid sequences of zinc fingers that bind sequences. For example, U.S. Patent Nos. 6,140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; U.S. Patent Application Publication Nos. 2002/0165356; 2004/0197889; 2007/0154989; 2007/0213269; and WO 98/53059 and 2003/016496.

  Exemplary selection methods, including phage display, interaction traps, hybrid selection and two-hybrid systems, are described in US Pat. Nos. 5,789,538; 5,925,523; 6,007,988; , 453; 6,140,466; 6,200,759; 6,242,568; 6,410,248; 6,733,970; 6,790,941; 7,029, 847 and 7,297,491, and US Patent Application Publication Nos. 2007/0009948 and 2007/0009962; International Publication No. WO 98/37186; International Publication No. 01/60970 and British Patent No. 2,338,237. Is disclosed.

  Enhanced binding specificity for zinc finger domains is described, for example, in US Pat. No. 6,794,136 (September 21, 2004). With respect to inter-finger linker sequences, further aspects of zinc finger modification are disclosed in US Pat. No. 6,479,626 and US Patent Application Publication No. 2003/0119023. See also Moore et al. (2001a) Proc. Natl. Acad. Sci. USA 98: 1432-1436; Moore et al. (2000b) Proc. Natl. Acad. Sci. USA 98: 1437-1441 and WO 01/53480.

  Transcriptional activation and repression domains are known in the art. See, for example, Science 269: 630 (1995). Exemplary transcriptional activation domains include p65, VP16 and VP64. Exemplary transcriptional repression domains include KRAB, KAP-1, MAD, FKHR, ERD, and SID. The functional domain from the nuclear hormone receptor serves as either an activator or suppressor, depending on the presence of the ligand. See also US Pat. No. 7,985,887.

Formulations, Kits and Routes of Administration As disclosed herein, therapeutic compositions comprising SB623 cells are also provided. Such compositions typically include SB623 cells and a pharmaceutically acceptable carrier. Additional active compounds can also be incorporated into the SB623 cell composition (see below).

  The therapeutic compositions disclosed herein are particularly useful for stimulating angiogenesis after the onset of stroke or other ischemic injury in a subject. Thus, a “therapeutically effective amount” of a composition comprising SB623 cells can be any amount that stimulates angiogenesis. Depending on body weight, route of administration, disease severity, etc., for example, the dosage may be about 100; 500; 1,000; 2,500; 5,000; 10,000; 20,000; 50,000; 100,000; 500,000; 1,000,000; 5,000,000 to 10,000,000 cells or more (or any integer value therebetween), for example, once a day The administration frequency can be twice a week, once a week, twice a month, once a month.

  Various pharmaceutical compositions and techniques for their preparation and use are known to those of skill in the art in light of the present disclosure. For a detailed list of suitable pharmacological compositions and techniques for their administration, texts such as Remington's Pharmaceutical Sciences, 17th ed. (. Eds) 1985;; Brunton et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics," McGraw-Hill, 2005 University of the Sciences in Philadelphia, "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins , 2005; and University of the Sciences in Philadelphia (eds.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkes ins, 2008.

  The cells described herein can be suspended in a physiologically compatible carrier for transplantation. As used herein, the term “physologically compatible carrier” is compatible with SB623 cells, compatible with any other component of the formulation, and not harmful to its recipients. Means carrier. Those skilled in the art are familiar with physiologically compatible carriers. Examples of suitable carriers include cell culture media (eg Eagle's minimum essential media), phosphate buffered saline, Hank's balanced salt solution +/− glucose (HBSS), and multiple electrolyte solutions, eg Plasma-Lyte ™ ) A (Baxter).

  The dose of SB623 cell suspension administered to a patient will vary depending on the site of implantation, therapeutic goal and the number of cells in the solution. Typically, the amount of cells administered to a patient is a therapeutically effective amount. As used herein, a “therapeutically effective amount” or “effective amount” is used to achieve treatment of a particular disease, ie, the amount of symptoms associated with the disorder and It means the number of transplanted cells needed to produce a reduction in severity. For example, in the case of a stroke, transplantation of a therapeutically effective amount of SB623 cells results in new blood vessel growth, blood vessel sprouting, and vessel branching, for example, in areas damaged by ischemia. The therapeutically effective amount will vary with the type and extent of ischemic injury and may also vary depending on the general condition of the subject.

  The disclosed therapeutic composition may further comprise a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, ie a carrier. These carriers can, for example, stabilize SB623 cells and / or facilitate survival of SB623 cells in the body. Each carrier should be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxycellulose , Ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; Glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; Examples include magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen removal water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic compatible substances used in pharmaceutical formulations. . Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, and colorants, mold release agents, coating agents, sweeteners, flavoring and fragrances, preservatives and antioxidants are also present in the composition. Can exist.

  Exemplary formulations include, but are not limited to, those suitable for parenteral administration, eg, pulmonary, intravenous, intraarterial, intraocular, intracranial, sub-meningial or subcutaneous administration, micelles, liposomes Or a drug release capsule (an active agent incorporated in a biocompatible coating designed for sustained release); an ingestible formulation; a formulation for topical use, eg eye drops, cream Ointments and gels; and other formulations such as inhalation, aerosols and sprays. The dosage of the disclosed compositions will vary according to the degree and severity of the treatment need, the activity of the administered composition, the overall health of the subject, and other considerations well known to those skilled in the art.

  In further embodiments, the compositions described herein are delivered locally to the ischemic injury site. Local delivery allows the composition to be delivered non-systemically, thereby reducing the burden of the composition on the body compared to systemic delivery. Such local delivery can be achieved, for example, by intracranial injection or through the use of various medical implant devices, including but not limited to stents and catheters, or by inhalation, phlebotomy, or surgery. Coating, implantation, embedding methods, and other methods of attaching desired agents to medical devices such as stents and catheters are established in the art and contemplated herein.

  Another aspect of the present disclosure relates to a kit for performing administration of SB623 cells to a subject, optionally in combination with other therapeutic agents. In one embodiment, the kit is formulated in a pharmaceutical carrier, optionally, for example, a pro-angiogenic agent (see below), and optionally in one or more individual pharmaceutical formulations, SB623. Contains a composition of cells.

Combination Therapy In certain embodiments, the SB623 cell composition is combined with other compositions comprising agents that stimulate angiogenesis (“pro-angiogenic agents”), eg, for treating stroke The compositions can be administered sequentially or simultaneously in any order, and as disclosed herein, the therapeutic composition comprises both SB623 cells and a pro-angiogenic agent. In a further embodiment, separate therapeutic compositions, one comprising SB623 cells and the other comprising a pro-angiogenic agent, can be administered to a subject individually or together.

  In certain embodiments, the angiogenesis-promoting agent is a protein (eg, fibroblast growth factor, platelet derived growth factor, transforming growth factor α, hepatocyte growth factor, vascular endothelial growth factor, sonic hedgehog, MAGP- 2, HIF-1, PR-39, RTEF-1, c-Myc, TFII, Egr-1, ETS-1) or a nucleic acid encoding such a protein. See, for example, Vincent et al. (2007) Gene Therapy 14: 781-789. In other embodiments, the pro-angiogenic agent can be a small RNA molecule (eg, siRNA, shRNA, microRNA) or a ribozyme that targets a nucleic acid encoding an angiogenesis inhibitor. In a further embodiment, the pro-angiogenic agent can be a triplex-forming nucleic acid that binds to a DNA sequence that regulates expression of a protein that inhibits pro-angiogenesis, eg, blocks transcription of the gene encoding the protein. .

  In a further embodiment, the pro-angiogenic agent is a transcription factor that activates the expression of a pro-angiogenic molecule (eg, a protein). Naturally occurring transcription factors (eg, HIF-1α) that regulate the expression of pro-angiogenic proteins are known. Furthermore, synthetic transcriptional regulatory proteins can be constructed by genetic engineering. For example, methods for designing zinc finger DNA binding domains that bind to sequences of interest and methods for fusing such zinc finger DNA binding domains with transcriptional activation and repression domains have been described. For example, US Pat. Nos. 6,534,261; 6,607,882; 6,785,613; 6,794,136; 6,824,978; 6,933,113; 6,979 539; 7,013,219; 7,177,766; 7,220,719; and 7,788,044. These methods can be used to synthesize non-naturally occurring proteins that activate transcription of any gene that encodes a pro-angiogenic protein. A synthetic zinc finger transcriptional activator of the vascular endothelial growth factor (VEGF) gene has also been described. See, for example, U.S. Patent Nos. 7,026,462; 7,067,317; 7,560,440; 7,605,140; and 8,071,564. Thus, non-natural (ie, synthetic) zinc finger proteins that activate transcription of the VEGF gene can be used in combination with SB623 cells to promote angiogenesis, for example, in the treatment of stroke.

  In further embodiments, natural or synthetic transcriptional regulatory proteins that inhibit transcription of anti-angiogenic molecules (eg, synthetic zinc finger transcriptional regulatory proteins) can be used as pro-angiogenic agents.

Example 1: Conditioned Medium MSC and SB623 cells were obtained and / or prepared as described. For example, U.S. Patent No. 7,682,825 (March 23, 2010) and U.S. Patent Application Publication No. 2010/0266554 (Oct. 21, 2010), 2010/0310529 (December 9, 2010). , 2011/0229442 (September 22, 2011), and 2011/0306137 (December 15, 2011); these disclosures refer to SB623 cells (in these references "neural progenitor cells" For the purposes of describing the preparation of “precursor cells” and “variably referred to as“ neural regenerating cells ”), they are incorporated by reference in their entirety. Cells were supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), 2 mM L-glutamine and 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA (both)), α-MEM (Mediatech) , Herndon, VA). MSC and SB623 cells typically expressed CD29, CD90, and CD105 and did not express CD31, CD34, or CD45 as measured by flow cytometry.

For use in the experiments described herein, frozen MSC and SB623 cells from the same human donor were thawed, replated in growth medium, and allowed to recover for about 1 week. To obtain the conditioned medium, the cells are grown to approximately 90% confluence (˜15,000 cells / cm ² ), the plate is washed once with phosphate buffered saline (PBS), and the medium is removed. This was then replaced with OptiMEM® medium (Invitrogen, Carlsbad, Calif.) To maintain the same cell density. Conditioned medium was collected after 72 hours. Frozen samples of conditioned media were slowly warmed to 37 ° C before use.

Example 2: Effect of SB623 cell secretory factor on HUVEC survival Cerebral ischemia can result in a loss of nutrient supply to the affected area. To determine whether SB623 cells and MSC-derived soluble factors have a recovery effect on nutrient-deficient endothelial cells, human umbilical vein endothelial cells (HUVEC) were cultured in serum and growth factor-deficient medium for 24 hours, Next, the conditioned medium (CM) derived from MSC or SB623 cells was used. The control medium remains serum and growth factor deficient medium without the addition of CM. HUVEC viability and proliferative capacity were then evaluated.

For these experiments, human umbilical vein endothelial cells were passed twice and then 7.5 × 10 ⁵ cells were coated with 0.1% gelatin in a T-75 flask in an EBM-2 / Seeded in ECGS medium (Endothelial Basal Medium-2 / Endothelial Cell Growth Supplement; Lonza, Walkersville, MD) and cultured for 24 hours. HUVEC monolayers were washed twice with warm PBS and incubated overnight at 37 ° C., 5% CO ² with 12 ml fresh EBM-2 medium. The effect of CM was to remove 6 ml of medium from each flask and replace it with 6 ml of fresh OptiMEM (control), 6 ml of MSC conditioned medium, or 6 ml of SB623 cell conditioned medium (as described in Example 1). Was then evaluated by replacing the conditioned medium prepared in 1). After 7 days, non-adherent and adherent cells were harvested, centrifuged at 1400 rpm for 5 minutes, and divided into three fractions (PL, Bcl-2 and Ki67) for subsequent staining analysis.

  Since dead cells are permeable to propidium iodide (PI), the cells were stained with PI to quantify cell death. Cells are stained with 5 μg / ml PI for 30 minutes at room temperature and flow cytometry collection and analysis is performed using the FL-2 logarithmic chamber of a BD FACSCalibur CellQuest program (BD Biosciences, San Jose, Calif.). It was. Three different human donor pairs were tested for this assay. The results are shown in FIG. In control HUVEC cultures maintained for 7 days in nutrient-deficient medium, more than 70% of the cells were positive for propidium iodide staining. Addition of either SB623 or MSC conditioned media significantly reduced the proportion of propidium iodide positive cells (p <0.05).

  These results indicate that both MSC-conditioned medium and SB623 cell-conditioned medium significantly reduce endothelial cell death caused by serum and growth factor deficiency (ie, reduce the number of propidium iodide positive HUVECs). .

  Bcl-2 is an anti-apoptotic protein that was originally identified as overexpressed in certain B cell lymphomas. Therefore, the cell fraction expressing Bcl-2 protein was measured as an indicator of their potential for apoptosis in serum / growth factor deficient HUVEC. For Bcl-2 measurements, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X100 for 1 hour. After permeabilization, cells were stained with fluorescein-conjugated anti-Bcl-2 for 1 hour on ice, then samples were washed, collected, and analyzed on the FL-1 channel of BD FACSCalibur. Cells subjected to fluorescein conjugated IgG were used as a negative control. For these assays, three different human donor pairs were tested.

  The results shown in FIG. 2 indicate that the presence of either MSC conditioned medium or SB623 cell conditioned medium significantly increased the Bcl-2 positive cell fraction in serum-deficient endothelial cell cultures.

  Conditioned media from NSC or SB623 cells reduced the number of dead cells (PI-positive) and increased the number of cells expressing anti-apoptotic Bcl-2 protein. Both MSC and SB623 cells Indicates that secreted factors that enhance endothelial cell survival.

Example 3: Effect of SB623 cell secretory factor on HUVEC proliferation Ki67 is a protein present in cells emanating from the G0 (resting) phase of the cell cycle; therefore, the level of Ki67 can be used as a measure of cell proliferation . The cell fraction expressing Ki67 protein was measured in HUVEC depleted of serum and growth factors and then cultured in conditioned medium from either MSC or SB623 cells.

  For Ki67 measurement, HUVEC were cultured and subjected to CM as described in Example 2. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X100 for 1 hour. After permeabilization, cells were stained with fluorescein-conjugated anti-Ki67 antibody for 1 hour on ice, after which samples were washed, collected, and analyzed on the FL-1 channel of BD FACSCalibur. Cells subjected to fluorescein conjugated IgG were used as a negative control. For these analyses, three different human donor pairs were tested.

  FIG. 3 shows the fraction of cells in the presence of conditioned medium from either MSC or SB623 cells in which deficient HUVEC cultures expressed increased Ki67 compared to control HUVEC not subjected to conditioned medium. Show that it brings. The conditioned medium from MSC or SB623 cells increased the number of cells expressing Ki67 protein associated with proliferation, indicating that MSC and SB623 cells secrete factors that enhance endothelial cell proliferation.

  The results shown in this and previous examples show that when these endothelial cells were cultured in MSC or SB623 cell conditioned medium for 7 days, HUVEC survival and proliferation compared to cultures in non-conditioned medium. Revealed a significant increase (p <0.05).

Example 4: Effect of SB623 cell secretory factor on tube formation by endothelial cells The HUVEC tube formation assay was used to test the ability of MSC and SB623 cells to elaborate factors that stimulate angiogenesis. For example, EJ Smith & CA Station, “Tube Formation Assays,” in Angiogenesis Assays-A Critical Application of Current Techniques, (Station, Lewis. John Wiley & Sons, Ltd. , West Sussex, UK, pp. 65-87, 2006; and Goodwin (2007) Microvasc. Res. 74: 172-183.

  HUVEC were passed 5 times through EBM-2 / ECGS medium and then transferred to α-MEM / 0.5% FBS / 2 mM glutamine / penicillin-streptomycin at a density of 1 × 10 5 cells / ml. After 24 hours, HUVECs were harvested using 0.25% trypsin-EDTA, washed and resuspended in α-MEM / 2 mM glutamine / penicillin-streptomycin at a density of 1 × 10 5 cells / ml. did. A mixture of either 75 μl HUVEC and 75 μl MSC or SB623 conditioned medium (Example 1), or 75 μl OptiMEM medium (negative control) was added to each well with 50 μl Reduced Growth Factor (RGF) -basement gel ( Invitrogen, Carlslsbad, CA) was added to each well of a 96-well plate that had been pretreated and the plate was incubated at 37 ° C. for 45 minutes. For this assay, MSCs were obtained from 3 different human donors and a portion of the MSC from each donor was converted to SB623 cells.

  After 16 hours, the culture was observed with a phase contrast microscope and photographs. The number of complete tubes (formed by the cells in contact) was quantified by an experimenter who was unaware of the group. A photograph showing the results from any of the three donors is shown in FIG. The results of the assay using MSC and SB623 cells from all three donors, summarized in FIG. 5, indicate that tube formation is strongly enhanced by conditioned media from either MSC or SB623 cells. Thus, MSC and SB623 cells secrete factors that promote angiogenesis.

Example 5: Effect of SB623 cell secretory factor on vascular elongation and branching Recovery of the vasculature after ischemic injury requires that the remaining endothelial cells receive signals that promote their migration and invasion. Such signals can arise from vascular smooth muscle cells, monocytes and / or macrophages, among others. An aortic ring assay was used to test the secretion of factors involved in vascular sprouting and branching. See, for example, Nicosia & Otinetti (1990) Lab. Invest. 63: 115-122 and Nicosia (2009) J. MoI. Cell. Mol. Med. 13: 4113-4136.

For preparation of the aortic ring, adult Sprague-Dawley rats were euthanized prior to dissection. The ends of the aorta were clamped with forceps, removed and transferred to ice-cold α-MEM / penicillin-streptomycin medium before removing the outer fat layer. Adipose-free aorta was washed twice with ice-cold EBM-2 / penicillin-streptomycin medium before being split into 1.0 mm thick rings. The aortic ring was then transferred to a plate containing EBM-2 / penicillin-streptomycin medium and incubated for 6 days at 37 ° C., 5% CO ₂ , with the medium being fresh EBM-2 / penicillin-streptomycin on day 3. The medium was replaced to deplete all endogenous rat angiogenic factors. At that time, the medium was replaced with α-MEM / penicillin-streptomycin medium and the culture continued for 24 hours.

On day 0 of the aortic ring assay (7 days after the start of culture), 50 μl of Reduced Growth Factor (RGF) basement gel was deposited in each well of a 24-well plate. Each aortic ring was placed in the middle of each gel-coated well and overlaid with an additional 25 μl of RGF basement gel. For gel clotting, after 30 minutes at 37 ° C./5% CO ₂ , 500 μl α-MEM / 2 mM glutamine / penicillin-streptomycin was added to each well and incubation continued for another 30 minutes. . Thereafter, 500 μl of either MSC or SB623-derived conditioned medium (Example 1) was added. As a negative control, 500 μl of OptiMEM medium was used instead of conditioned medium.

  To assess the angiogenic activity of MSC and SB623-derived factors, phase contrast photographs were taken on day 10 and the results were counted by vascular extension and branching by a group-inexperienced experimenter. Quantified. New blood vessel growth was quantified by measuring the number of blood vessels extending from the annulus; vascular bifurcation was quantified by measuring the number of branch points present in the blood vessel extending from the aortic annulus.

  Representative results from the 10 day sample are shown in FIG. 6, and results from 7 sets of 10 day cultures are summarized and quantified in FIG. FIG. 7A shows that conditioned media from both MSC and SB623 cells stimulated an increase in the number of newly sprouted blood vessels and the degree of branching compared to control aortic rings. Furthermore, a significant increase in vascular branching was observed in SB623 cell acclimation compared to either rings grown in MSC conditioned medium (FIG. 6B, FIG. 7A) or rings cultured in non-conditioned medium (FIG. 6A, FIG. 7A). Observation was performed on the rings cultured in the medium (FIG. 6C, FIG. 7A). These results indicate that MSC and SB623 cells secrete factors that promote vascular sprouting and vascular branching. In particular, SB623 cells secrete factors that greatly promote vascular bifurcation (see FIG. 7B).

  The data presented in the previous examples show that SB623 cell secreted soluble factor promotes several aspects of angiogenesis and contributes to recovery in the damaged brain.

Example 6: Statistics For each experiment (3-4 wells / group), the mean values were: (1) treatment conditions for each cell type (conditioned media from MSC or SB623 cells; one value per human donor tested) and ( 2) Obtained for the untreated group (one value for each test). For statistical comparisons (SigmaStat, SysstatSoftware, Chicago, IL), each of these values was used, and comparisons were made between the following groups (1) Control (unconditioned medium; n = 3), (2) MSC conditioned medium ( n = 3-5); and (3) SB623 cell conditioned medium (3-5) using one way ANOVA. Additional pair-wise comparisons were made using the Tukey method. An alpha value of 0.05 was used to determine if the means were significantly different.

Example 7: Identification of angiogenic factors secreted by MSC and SB623 cells The levels of specific cytokines and trophic factors in MSC and SB623 cells were measured. To obtain conditioned medium, MSC and SB623 cells were cultured in growth medium to 90% confluence (˜15,000 cells / cm ² ) at which point the medium was removed and the cells were washed in PBS. And OptiMEM® medium (Invitrogen, Carlsbad, Calif.) Was added to obtain a concentration of ˜150,000 cells / ml. Conditioned medium was harvested after 72 hours and assayed using Quantobody® Human Analysis Assay 1 (RayBiotech, Norcross, GA) according to instructions. For each source of MSC, a portion of the cells were cultured directly as MSC and a portion was converted to SB623 cells. Therefore, cultures of MSCs from specific donors and cultures of SB623 cells made from these MSCs are referred to as corresponding “donor pairs”. In this experiment, four donor pairs were assayed. The concentration expressed as the concentration of protein was normalized to the number of cells present in the culture when the conditioned medium was collected. FIG. 8 shows the results for angiogenin, ANG-2, HB-EGF and PIGF by the donor. FIG. 9 also shows the results for these four factors and the other six by the donor and highlights the large amount of VEGF produced by MSC and SB623 cells.

  Table 1 shows the average protein level among the 4 donor pairs for the 10 tested factors. Although the levels of secreted trophic factors varied between different donors (eg, as shown in FIGS. 8 and 9), the levels of the four factors (Angiogenin, Angiopoietin-2, HB-EGF and PIGF) are: Consistently different between MSC and SB623 cells. Angiogenin, ANG-2, and HB-EGF were more highly expressed by SB623 cells, but higher concentrations of PIGF were produced by MSC.

Abbreviations are as follows: ANG-2: Angiopoietin-2; EGF: epidermal growth factor; bFGF: basic fibroblast growth factor / fibroblast growth factor 2; HB-EGF: heparin-binding epidermal growth factor-like growth factor HGF: hepatocyte growth factor; PDGF-BB: platelet derived growth factor-BB; PIGF: placental growth factor; VEGF: vascular endothelial growth factor. Numbers refer to cytokine levels expressed as pg / ml / 10 ⁶ cells. “AVG” means the mean value from 4 sources of MSC and 4 sources of SB623 cells from which conditioned media was obtained. “SD” means standard deviation. “-” Indicates that the level, if any, was below the limit of detection in the assay. “N / A” indicates “not applicable”.

Example 8: Effect of VEGF signaling inhibition on HUVEC survival and proliferation Considering the large amount of VEGF secreted by both MSC and SB623 cells, the contribution of VEGF to the pro-angiogenic activity of MSC and SB623 conditioned media is Tested using inhibitors of VEGF signaling. SU5416 (VEGFR2 kinase inhibitor III, EMD Millipore, Billerica, MA) is down-regulated by VEGF receptor 2 (Flk-1), to a lesser extent by VEGF receptor 1 (Flt-1) and other receptor tyrosine kinases. Blocks signal transduction, thereby inhibiting angiogenesis.

  HUVEC viability assays (propidium iodide uptake and Bcl-2 expression) were performed in the presence and absence of 50 nM SU5416 for two batches of SB623 cell conditioned medium, cells were replaced for 7 days prior to the assay. The procedure was as described in Example 2, except that the cells were cultured for 5 days. Inhibitors were added to the culture 30 minutes prior to the addition of CM. Since higher concentrations of SU5416 can inhibit other receptor tyrosine kinases than VEGFR2, this SU5426 concentration can be reduced to VEGFR2 signaling (but, for example, not transmitted by PDGF receptor, EGF receptor, or Flt3). Was selected to inhibit. The results shown in FIGS. 10A and 10B show that more cells incorporated PI when HUVECs were cultured in SB623 conditioned medium and SU5416 than when they were cultured in SB623 cell conditioned medium alone. (FIG. 10A) and shows that the cells express less anti-apoptotic protein (FIG. 10B). Thus, inhibition of VEGF receptor activity partially reduces the positive impact of SB623 cell conditioned media on HUVEC viability, indicating the role of VEGF protein in these effects.

  The effect of VEGF receptor inhibitors on stimulation of HUVEC proliferation by SB623 cellular factor was also evaluated. An assay for Ki67 expression is shown in Example 3 except that 50 nM SU5416 was added to the medium 30 minutes before the addition of conditioned medium and the cells were cultured for 5 days instead of 7 days prior to the assay. Performed as described. The results shown in FIG. 11 and averaged from two donors indicate that the increase in HUVEC proliferation observed in the presence of conditioned media from SB623 cells was partially reversed by inhibition of VEGFR2.

  These results point to a role for VEGF in addition to other SB623 cell-derived factors in the survival promoting and growth promoting activities of MSC and SB623 cell conditioned media.

Example 9: Effect of VEGF signaling inhibitors on tube formation by human umbilical vein endothelial cells (HUVEC) HUVEC tube formation assays with conditioned media from MSC and SB623 cells were performed in the presence and absence of the VEGF2 receptor inhibitor SU5416. Performed as described in Example 4 in the presence. Cells cultured in the absence of conditioned medium were used as negative controls; and cells cultured in the presence of VEGF (10 ng / ml) were used as positive controls. The results shown in FIG. 12 show that VEGF, MSC conditioned medium and SB623 cell conditioned medium all promoted tube formation, whereas the VEGF2 inhibitor SU5416 reduced stimulation of tube formation by all these agents. Indicates.

  Quantification of tube formation was performed as described in Example 4 on HUVECs subjected to SB623 cell conditioned medium at 16 and 40 hours after seeding, in the presence and absence of SU5416. The results shown in FIG. 13 show that at both time points, inhibition of VEGFR2 completely reverses the positive effect of CM on tube formation.

Example 10 Effect of VEGF Signaling Inhibition on Vascular Elongation and Bifurcation in the Aortic Ring Assay An aortic ring angiogenesis assay is performed in one batch of SB623 cell conditioned medium in the presence and absence of 50 nM SU5416. The procedure was as described in 5. Inhibitors were added to the cultures 30 minutes before the addition of CM, and rings were assayed after 10 days in culture. Results show that vascular elongation and branching from aortic ring cultures in SB623 cell conditioned medium (FIG. 14, left and middle panel comparison) in the presence of the VEGF receptor inhibitor SU541 (FIG. 14, middle and right). (Comparison of panel) shows that it was reduced. These results provide further evidence for the role of VEGF in the pro-angiogenic activity of SB623 cell conditioned media.

  While the results obtained using VEGF receptor inhibitors (above) confirmed the importance of VEGF for these processes (especially tube formation, vessel elongation and vessel branching), MSC and SB623 cell conditioned media It does not exclude the involvement of additional factors (other than VEGF) in the pro-angiogenic activity of.

Claims (22)

Ischemic injury in a subject by delivery of one or more angiogenic factors selected from the group consisting of angiogenin, angiopoietin-2 , basic fibroblast growth factor, leptin, platelet derived growth factor-BB, and placental growth factor Use of a cell in the manufacture of a medicament for use in repair of a cell, wherein said cell is:
(A) preparing a culture of mesenchymal stem cells;
(B) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length Notch protein;
Use obtained by (c) selecting cells comprising the polynucleotide of step (b) and (d) further culturing the selected cells of step (c) without selection.   The repair of ischemic damage is selected from the group consisting of promoting angiogenesis, preventing endothelial cell death, improving endothelial cell survival, stimulating endothelial cell proliferation, and promoting vascular bifurcation. use.   Use according to claim 1 or 2, wherein the ischemic damage occurs in the central nervous system.   4. Use according to claim 3, wherein the ischemic injury occurs in the brain.   The use according to any one of claims 1 to 4, wherein the medicament further comprises an angiogenesis promoter.   The use according to claim 5, wherein the angiogenesis-promoting agent is a polypeptide.   The use according to claim 5, wherein the angiogenesis-promoting agent is a nucleic acid encoding a polypeptide.   The use according to claim 6 or 7, wherein the polypeptide is a transcription factor that activates expression of a pro-angiogenic protein.   Use according to claim 8, wherein the pro-angiogenic protein is vascular endothelial growth factor (VEGF).   The use according to claim 8 or 9, wherein the transcription factor is a non-natural zinc finger protein that activates transcription of a VEGF gene.   11. Use according to any one of claims 5 to 10, wherein the ischemic injury is a stroke. Use of a cell in the manufacture of a medicament for supplying an angiogenic factor to a subject, wherein said angiogenic factor is angiogenin, angiopoietin-2 , basic fibroblast growth factor, leptin, platelet derived growth factor -BB, and one or more selected from the group consisting of placental growth factor; wherein the cell is:
(A) preparing a culture of mesenchymal stem cells;
(B) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length Notch protein;
Use obtained by (c) selecting cells comprising the polynucleotide of step (b) and (d) further culturing the selected cells of step (c) without selection.   13. The angiogenic factor is provided for one or more of promoting angiogenesis, preventing endothelial cell death, improving endothelial cell survival, stimulating endothelial cell proliferation, or promoting vessel branching. Use of.   14. Use according to claim 12 or 13, wherein the angiogenic factor is supplied to the central nervous system.   15. Use according to claim 14, wherein the angiogenic factor is supplied to the brain.   The use according to any one of claims 12 to 15, wherein the medicament further comprises an angiogenesis promoter.   The use according to claim 16, wherein the angiogenesis-promoting agent is a polypeptide.   The use according to claim 16, wherein the angiogenesis-promoting agent is a nucleic acid encoding a polypeptide.   The use according to claim 17 or 18, wherein the polypeptide is a transcription factor that activates the expression of a pro-angiogenic protein.   20. Use according to claim 19, wherein the pro-angiogenic protein is vascular endothelial growth factor (VEGF).   21. Use according to claim 19 or 20, wherein the transcription factor is a non-natural zinc finger protein that activates transcription of the VEGF gene.   The use according to any one of claims 16 to 21, wherein the angiogenic factor is provided for the treatment of stroke.